A new recombinant human bone morphogenetic protein-2 carrier for bone regeneration.

A gelatin sponge was formed by foaming and heat treating a gelatin solution, followed by coating the solid with poly(D,L-lactic-co-glycolic acid) to reinforce the gelatin framework. This sponge was tested for its suitability as a biodegradable porous, recombinant human bone morphogenetic protein (rhBMP)-2 carrier. Incorporation of rhBMP-2 into the sponge was closely related to its bulk density of gelatin sponge. The calcium content in the sponges, as assessed by an ectopic bone formation assay in rats, increased with the increasing sponge bulk density. Histologic and peripheral quantitative computed tomography analysis of implants in this ectopic assay system revealed cell growth throughout the carrier in 4 weeks after implantation regardless gelatin bulk density. The carrier containing rhBMP-2 maintained its three-dimensional structure after implantation; the carrier resisted collapse caused by soft tissue pressure during rapid bone formation as assessed by soft X-ray photographs. These results indicate that this newly developed sponge has excellent carrier characteristics to introduce rhBMP-2 into areas needed for bone regeneration.

Laminin and estradiol regulation of the plasminogen-activator system in MCF-7 breast-carcinoma cells.

We have investigated the effects of laminin, on the plasminogen-activator system of MCF-7 breast-carcinoma cells. MCF-7 cells were incubated on plastic or laminin-coated wells, and medium and cell lysate aliquots were assayed for tissue-type (tPA) and urokinase-type plasminogen activator (uPA) by a chromogenic assay in combination with anti-uPA antibodies. Cells cultured on laminin displayed a 5-fold increase in tPA activity and a 2-fold decrease in uPA activity relative to cells on plastic. These effects could be mimicked by laminin fragment P1 but not by collagen I or fibronectin. tPA activity of cells treated with estradiol (10 nM) was 3-fold higher, that of cells on laminin treated with estradiol was 15-fold higher, than that of control. Northern-blot analysis showed that tPA mRNA levels were up-regulated by estradiol and laminin, whereas PAI-1 mRNA levels were down-regulated by laminin and not affected by E2. Concomitant treatment with laminin and estradiol, decreased PAI-1 mRNA and increased tPA mRNA levels, accounting for the synergistic increase in tPA activity. Laminin exerted only a modest (approx. 2-fold) inhibitory effect on uPA mRNA levels. In the breast-carcinoma cell line MDA-MB-231, down-regulation of PAI-1 and uPA mRNA by laminin was not observed. Adhesion assays indicated that alpha2beta1 is the predominant receptor for laminin in MCF-7 cells. MDA-MB-231 cells expressed alpha2 (54%) but this integrin is not used as a laminin receptor. These results support a role for alpha2beta1 in mediating interactions of MCF-7 with LN.

Differential expression of fibronectin receptors during myeloid differentiation and in marrow- and peripheral blood-derived leukemic cells.

A 125I-labeled 120-kDa fibronectin fragment (FN120) containing the RGD binding site was employed to assess FN120 receptor levels in control and dimethylsulfoxide (DMSO)-differentiated HL60 cells, as well as in leukemic peripheral and bone marrow blast cells from acute lymphoid (ALL) and myeloid (AML) patients. Fibronectin CS1 fragment receptor alpha 4 (VLA4-alpha) and RGD-dependent alpha 5 integrin subunits (VLA5-alpha) were characterized by specific monoclonal antibodies (MoAb). HL60 cells, induced along the granulocytic pathway with DMSO, displayed low FN120 binding level densities (36,070 +/- 5142 sites/cell (s/c) vs. 19,780 +/- 4564 s/c, P < 0.005), respectively, for untreated and treated cells) together with decreased VLA5-alpha expression. Granulocytes displayed low levels of FN120 receptors (3167 +/- 1165 s/c) with weak VLA5-alpha expression and absence of VLA4-alpha. Normal lymphocytes displayed 17,670 +/- 8,705 s/c FN120 receptors and VLA4-alpha and VLA5-alpha. The mean FN120 binding levels and mean VLA5-alpha expression were lower in peripheral blast cells, both in ALL and AML, than in the bone marrow leukemic cells. VLA4-alpha remained the same irrespective of cell localization. FN120 binding sites and differential expression of VLA4-alpha and VLA5-alpha integrin molecules on hemopoietic cells could be related to lineage characteristics or cell type distribution within hemopoietic tissue.

Regulation of extracellular matrix-degrading proteases

Extracellular matrix-degrading enzymes have a very important role in many normal and pathological processes. Members of the matrix metalloproteinase and plasminogen activator families are the major modulators of extracellular matrix degradation. Here, we discuss some topics about these enzymes giving special attention to the transcriptional and extracellular regulation of their expression.

Sex steroids induced up-regulation of 1,25-(OH)2 vitamin D3 receptors in T 47D breast cancer cells.

There is evidence indicating that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through binding to its specific receptor (VDR) exerts an antiproliferative effect on breast cancer cells. Considering the importance of receptor regulation in modulating the target cell responsiveness to hormones, the effect of dihydrotestosterone (DHT) and estradiol-17 beta (E2) on the regulation of VDR number was investigated in T 47D human breast cancer cells that also express androgen and estrogen (ER) receptors. T 47D cells were grown in RPMI medium containing 10% charcoal-treated fetal calf serum and the receptor content was determined in cells at confluence. Whole cell binding studies confirmed the presence of highly specific, saturable (4.01 +/- 1.82 fmol/10(6) cells), high affinity (Kd = 0.079 +/- 0.058 x 10(-9) M) 1,25(OH)2D3 receptors in control cells. Exposure to 10(-7) M DHT for 72 h resulted in a significant increase in VDR levels. Similar results were obtained with 10(-7) M E2. DHT- and E2-induced up-regulation was completely suppressed by 10(-6) M tamoxifen (TAM) addition but unaffected by 10(-6) M flutamide. TAM treatment alone produced a significant dose-dependent increase in VDR content, that was maximal at 10(-6) M. Our data strongly suggest, for the first time, an up-regulation of VDR by DHT and E2 via an ER-mediated mechanism.

Effects of androgens on proliferation and progesterone receptor levels in T47D human breast cancer cells.

The effects of estradiol (E2), dihydrotestosterone (DHT) and dehydro-3-epiandrosterone (DHEA) on proliferation and progesterone receptor induction were studied in a breast cancer cell line (T47D) expressing estrogen, androgen, and progesterone receptors. A significant enhancement of growth and progesterone receptor expression was observed after treatment with E2 and DHEA, which was antagonized by the antiestrogen tamoxifen and not altered by the antiandrogen flutamide, supporting the involvement of estrogen receptors. When cells were treated with either E2 or DHEA, transforming growth factor-alpha mRNA was induced. DHT treatment did not alter growth but was effective in stimulating androgen receptors and down-regulating progesterone receptors.

Vitamin D3 binding activity during leukemic cell differentiation.

In this paper we report that differentiation of the human promyelocytic leukemia cell line, HL60, along the myelocytic pathway, induced by retinoic acid (RA), or monocytic pathway, induced by phorbol-myristate acetate (PMA) and gamma interferon (IFN), was accompanied by a significant decline in 1,25-dihydroxycholecalciferol (1,25(OH)2D3) binding (control: 30.3 +/- 3.0 fM/10(6) cells; RA treated: 6.8 + 2.5 fM/10(6) cells; PMA treated: 12.3 +/- 6.7 fM/10(6) cells and IFN treated: 16.0 +/- 5.0 fM/10(6) cells). When differentiation and proliferation were uncoupled, by incubation with IFN or by inhibition of proliferation by cell density saturation, 1,25(OH)2D3 binding was better related to differentiation than to proliferation. Additionally we have compared 1,25(OH)2D3 binding levels in blasts from acute lymphocytic leukemia (ALL) patients (25.4 +/- 18.1 fM/10(6) cells) and normal, mature lymphocytes (10.6 +/- 2.1 fM/10(6) cells). Receptor binding was significantly higher (p < 0.05) in the immature blasts. Our data suggest that 1,25(OH)2D3 receptor levels could be considered a marker of functional immaturity, in these cells.

Increased expression of laminin receptors during myeloid differentiation.

Variation of laminin receptor levels (LNR) during myeloid-cell differentiation and in acute leukemia were investigated by 125I-laminin-binding determination during HL60 cell differentiation and in cells of patients with different types of leukemia, characterized according to the FAB classification. LNR levels in HL60 cells increased during differentiation, being significantly higher in cells exposed to phorbol myristate acetate (PMA) and ethanol (55,391 +/- 27,845 and 29,314 +/- 6,435 sites/cell respectively) as compared with HL60 controls (8,549 +/- 4,000 sites/cell). The control cells do not adhere to laminin-coated surfaces, but differentiation with PMA results in their rapid adherence on this substratum. Short treatment with PMA does not increase the number of adherent cells or the receptor expression. Granulocytes also presented equally high LNR concentration (29,739 +/- 13,516 sites/cell). The lymphoid cells (lymphocyte, acute lymphoid leukemia and chronic lymphocytic leukemia) shared low LNR numbers (less than 6,500 sites/cell). Myeloid cells displayed a wide range of LN receptors with higher levels being associated with the more differentiated FAB subgroups. 125I-laminin binding to lymphoid or myeloid leukemic cells was mainly inhibited by P1 fragments, whereas granulocytes and differentiated HL60 cells displayed a dual binding pattern for laminin fragments P1 and E8. These results were confirmed by assays using 125I-labelled P1 and E8 fragments. We conclude that magnitude of LNR levels and variation in expression of P1 and E8 receptors appear to be linked to lineage and maturation status in hematopoietic cells.

Effects of steroids on laminin-binding integrins in a human melanoma cell line.

The MEL-85 human melanoma cell line was used to investigate the effects of both estradiol and dexamethasone on expression of laminin (LM) receptors and cell adhesion capacity. Immunoblotting of eluates from whole-cell extracts applied to LM Sepharose indicates the presence of an LM-binding protein of 116-130 kDa that reacted with an anti-beta 1 integrin antibody, suggesting that the putative LM receptor of MEL-85 cells is a member of the integrin family. Analysis of 125I-LM binding to whole cells indicates the existence of low-affinity components which display positive co-operativity. LM-fragment-8 competes for this binding to the same extent as unlabelled LM (75%), while fragment PI is inactive and fibronectin (FN) competes by about 30% only. Binding of labelled fragment-8 exhibits a pattern similar to that of intact LM. Cell adhesion to substrates coated with LM and LM fragments closely parallels binding to cells in suspension. MEL-85 cells were estradiol-receptor-negative. Estradiol treatment did not stimulate LM receptor levels or attachment to LM. Growth rate also remained unaltered. To characterize the glucocorticoid dependence of MEL-85 cells, we first established the presence of glucocorticoid receptors and an inhibitory effect on growth rate. Dexamethasone treatment resulted in marked enhancement of adhesion to LM, without altering LM receptor number or affinity. In addition, dexamethasone changed the morphology of MEL-85 cells in conjunction with higher LM expression as evaluated by immunofluorescence.

Estrogen control of lactate dehydrogenase isoenzyme-5 in human breast cancer.

Determinations of estradiol receptor (ER), progesterone receptor (PR), total lactate dehydrogenase activity (LDH) and electrophoretic separation of LDH isoenzymes were performed in cytosols from 118 samples of primary infiltrating ductal mammary carcinoma. ER + PR+ tumors demonstrated a significant increase in the proportion of the LDH muscle-type isoenzyme (LDH5) as compared to ER-PR- samples (p less than 0.002). Tumors lacking one of the receptors presented intermediate LDH5 percentages. As total LDH activity bore no relation to either the presence or absence of receptors, the increased proportion represents an absolute elevation of LDH5, suggesting that LDH5 may be a promising hormone-dependence marker. As an in vitro model to study whether LDH5 was induced by estradiol via ER, we have used two human breast cancer cell lines, MCF-7 and T47D. In both cell lines LDH5 was the sole isoenzyme. Total ER and PR have been determined by a whole-cell method. In MCF-7 cells (with high ER levels), after incubation with 10(-10) M estradiol, maximal induction of LDH had already been achieved. In relation to T47D (low ER levels) estradiol did not evoke an induction of LDH5 at any concentration examined. In MCF-7 cells, the level of LDH5 induction paralleled processing of ER. The processing effect was rapid, beginning within 5 min of estradiol addition, and was completed within 1 hr. With 10(-6) M tamoxifen, LDH5 induction was suppressed and this effect was reversed by estradiol. Such antiestrogenic effects of tamoxifen on LDH5 have not been observed in T47D cells. Agonistic effects of low doses of tamoxifen on LDH5 were not observed. Our studies suggest that estrogen stimulation of LDH5 involves ER.

Construction of a cDNA clone corresponding to mouse alpha 1(IV) procollagen.

A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody.

Purification and characterization of procollagen mRNA from a heavy polysomal fraction.

A method is described for the purification of intact procollagen mRNA from heavy polysomal fractions isolated from 10 day-old chick embryos. Procollagen mRNA activity was characterized by gel electrophoresis under denaturing conditions and by its activity in in vitro cell-free translation systems derived from wheat germ and rabbit reticulocytes. 7-Methyl-guanosine monophosphate inhibited the translation of procollagen mRNA and globin mRNA to the same extent. Collagenase-sensitive polypeptides synthesized displayed the same electrophoretical mobilities as marker procollagen chains. The purity of the mRNA was 78% on the basis of ROt plot analysis of hybridization with its corresponding cDNA, using globin cDNA as the reference for 100%.

Reiteration frequency of procollagen genes in the guinea pig genome. Collagen genes are not amplified during granuloma fibroblasts differentiation.

Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen 125I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot 1/2 800-900 mol . s . 1-1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11-13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.

Procollagen biosynthesis and processing in guinea pig fibroblast culture medium.

1. Collagen biosynthesis and processing by primary cultures (2nd subculture) of guinea pig embryo fibroblasts were studied. Collagen represents about 15% of the protein synthesized and extruded by these cells under the culture conditions employed. 2. After incubating the cells with [3H]-proline for varying periods of time, the labelled products from the cell layer and the medium were analyzed by polyacrylamide gel electrophoresis. 3. Fluorography of the corresponding gels showed that the cell layer contained only mature alpha 1 (I) and alpha 2 chains, while the medium contained five extra bands; four with mobilities between beta-dimers and alpha 1 (I), and one migrating between alpha 1 (I) and alpha 2. These molecules were identified as predominantly type-I collagen precursors and represent about 96% of the collagen synthesized by the fibroblasts. 4. Kinetic studies of procollagen processing after both short- and long-term labelling periods showed that removal of amino- and carboxy-terminal peptide extensions occurred independently.